Reed-Sternberg cells in Hodgkin's lymphoma present features of cellular senescence.

Hodgkin's Lymphoma (HL) is one of the most prevailing malignancies in young adults. Reed-Sternberg (RS) cells in HL have distinctive large cell morphology, are characteristic of the disease and their presence is essential for diagnosis. Enlarged cells are one of the hallmarks of senescence, but whether RS cells are senescent has not been previously investigated. Here we show that RS cells have characteristics of senescent cells; RS cells in HL biopsies specifically express the senescence markers and cell cycle inhibitors p21Cip1 and p16INK4a and are negative for the proliferation marker Ki-67, suggesting that these cells have ceased to proliferate. Moreover, the RS-like cells in HL lines, stained specifically for senescence-associated ß-galactosidase (SA-ß-gal). Oxidative stress promoted senescence in these cells as demonstrated by their staining for p21Cip1, p16INK4a, p53 and γH2AX. Senescent cells produce copious amounts of inflammatory cytokines termed 'senescence-associated secretory phenotype' (SASP), primarily regulated by Nuclear Factor κB (NF-κB). Indeed, we show that NF-κB activity and NF-κB-dependent cytokines production (e.g., IL-6, TNF-α, GM-CSF) were elevated in RS-like cells. Furthermore, NF-κB inhibitors, JSH-23 and curcumin reduced IL-6 secretion from RS-like cells. Thus, defining RS cells as senescent offers new insights on the origin of the proinflammatory microenvironment in HL.

An imperfect correlation between DNA replication activity of Epstein-Barr virus nuclear antigen 1 (EBNA1) and binding to the nuclear import receptor, Rch1/importin alpha.

Epstein-Barr virus (EBV) replicates as a stable multicopy episome in latently infected mammalian cells. Latent cycle DNA replication requires only two viral elements, the cis-acting origin of plasmid replication (oriP) and the trans-acting origin binding protein (EBNA1). EBNA1 binds multiple recognition sites in oriP, but has not other enzymatic activities associated with replication functions. To identify human cellular proteins that mediate EBNA1 function, we designed a one-hybrid assay in yeast to select for proteins that bind to EBNA1 when bound to criP in vivo. A human cDNA encoding the Rch1/hSRP1 alpha/ importin alpha protein was isolated and shown to bind to full-length EBNA1, but not to an amino terminal deletion mutant of EBNA1 when bound to oriP in yeast. The interaction of EBNA1 with Rch1 was confirmed biochemically by coimmunoprecipitation from nuclear extracts and by direct binding of recombinant proteins in vitro. Internal deletion mutations in EBNA1 which compromised DNA replication activity were similarly reduced for binding to Rch1. Mutations with no effect on DNA replication activity were similarly unaffected for Rch1 binding. Rch1/importin alpha has been shown to bind to the nuclear localization sequence (NLS) of several proteins and stimulate nuclear import. A substitution mutation in the EBNA1 nuclear localization sequence reduced Rch1 binding, but had no effect on DNA replication function, indicating that Rch1 binding affinity does not correspond precisely with replication activity. Nevertheless, the identification of a stable interaction between Rch1 and EBNA1 at the origin of viral DNA replication raises the intriguing possibility that Rch1 contributes to the nuclear functions of EBNA1.

Intestinal apolipoprotein AI gene transcription is regulated by multiple distinct DNA elements and is synergistically activated by the orphan nuclear receptor, hepatocyte nuclear factor 4.

We have used apolipoprotein genes to investigate the signal transduction mechanisms involved in the control of intestinal specific gene expression. The human apoAI, apoCIII, and apoAIV genes are tandemly organized within a 15-kb DNA segment and are expressed predominantly in the liver and intestine. Transient transfection of various human apoAI gene plasmid constructs into human hepatoma (HepG2) and colon carcinoma (Caco-2) cells showed that apoAI gene transcription is under the control of two separate and distinct cell-specific promoters. The region between nucleotides -192 and -41 is essential for expression in HepG2 cells, whereas the region from -595 to -192 is essential for expression in Caco-2 cells. A third 0.6 kb DNA fragment in the apoCIII gene promoter region, approximately 5 kb down-stream from the human apoAI gene, enhances transcription mediated by either of these two tissue-specific apoAI promoters. In Caco-2 cells, expression of the apoAI gene and activation by the distal enhancer required the presence of a nuclear hormone receptor response element (NHRRE) located in the -214 to -192 apoAI promoter region. Overexpression of the orphan receptor hepatocyte nuclear factor 4 (HNF-4), which binds to the NHRRE, dramatically stimulates apoAI gene expression in Caco-2 cells but not in HepG2 cells. Maximal stimulation of transcription by HNF-4 in Caco-2 cells required the presence of both the intestinal specific promoter, the NHRRE, and distal enhancer elements. Transactivation by HNF-4 thus appears to result from functional synergy between the NHRRE binding HNF-4 and distal DNA elements containing intestinal-specific DNA binding activities. The apoAI gene provides a model system to define the mechanism(s) governing intestinal cell specific gene regulation and the role of nuclear hormone receptors in the establishment and regulation of enterocytic gene transcription.

The human myeloid cell nuclear differentiation antigen gene is one of at least two related interferon-inducible genes located on chromosome 1q that are expressed specifically in hematopoietic cells.

We have previously shown that the human myeloid cell nuclear differentiation antigen (MNDA) is expressed at both the antigen and mRNA levels specifically in human monocytes and granulocytes and earlier stage cells in the myeloid lineage. A 200 amino acid region of the MNDA is strikingly similar to a region in the proteins encoded by a family of interferon-inducible mouse genes, designated Ifi-201, Ifi-202, Ifi-203, etc, that are not regulated in a cell- or tissue-specific fashion. However, a new member of the Ifi-200 gene family, D3, is induced in mouse mononuclear phagocytes but not in fibroblasts by interferon. The same 200 amino acid region, duplicated in the mouse Ifi-200 gene family, is also repeated in the recently characterized human IFI 16 gene that is constitutively expressed specifically in lymphoid cells and is induced in myeloid cells by interferon gamma. The 1.8-kb MNDA mRNA, which contains an interferon-stimulated response element in the 5' untranslated region, was significantly upregulated in human monocytes exposed to interferon alpha. Characterization of the MNDA gene showed that it is a single-copy gene and localized to human chromosome 1q 21-22 within the large linkage group conserved between mouse and human that contains the Ifi-200 gene family. The IFI 16 gene is also located on human chromosome 1q. Our observations are consistent with the proposal that the MNDA is a member of a cluster of related human interferon-regulated genes, similar to the mouse Ifi-200 gene family. In addition, one mouse gene in the Ifi-200 gene family and the human MNDA and IFI 16 genes show expression and/or regulation restricted to cells of the hematopoietic system, suggesting that these genes participate in blood cell-specific responses to interferons.

Isolation and characterization of a cDNA clone for the CCAAT transcription factor EFIA reveals a novel structural motif.

Enhancer factor I (EFI) is a trans-acting factor which binds to the Rous sarcoma virus long terminal repeat enhancer and promoter at two inverted CCAAT-box motifs. We demonstrate that two forms of EFI DNA binding activity exist in nuclear extracts of avian cells. One form requires two heterologous components (EFIA)(EFIB) for high affinity, specific DNA binding activity, whereas a second form is not dependent on EFIB for binding and may be composed solely of EFIA, perhaps as a multimer. Both forms give rise to the same mobility shift in gel retardation assays, but the two forms can be separated chromatographically under buffer conditions which stabilize the two DNA binding activities. A cDNA for EFIA has been isolated from a rat liver cDNA expression library. The 1489-base pair EFIA cDNA encodes a 322-amino acid protein which is nearly identical to two previously described human DNA binding proteins. These are dbpB, a DNA binding protein of unknown specificity which binds to the epidermal growth factor receptor enhancer and c-erbB-2 gene promoter (Sakura, H., Maekawa, T., Imamoto, F., Yasuda, K., and Ishii, S. (1988) Gene (Amst.) 73, 499-507), and YB-1, a protein which recognizes the Y-box (inverted CCAAT motif) of the HLA-DR alpha chain gene (Didier, D. K., Schiffenbauer, J., Woulfe, S. L., Zacheis, M., and Schwartz, B. D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7322-7326). EFIA/dbpB/YB-1 share a highly conserved region of 100 amino acids with dbpA, another protein identified by Sakura et al. (1988) which binds to the epidermal growth factor receptor enhancer and c-erbB-2 gene promoter, and with two Xenopus CCAAT binding proteins, FRG Y1 and FRG Y2 (Tafuri, S. R., and Wolffe, A. P. (1990) Proc. Natl. Acad. Sci. U. S. A., in press). This highly conserved domain among all six proteins is presumed to represent or contain a DNA binding domain for the CCAAT motif. In addition, we note that the EFIA/dbpB/YB-1 polypeptide contains a novel arrangement of alternating clusters of positively and negatively charged amino acids not yet reported for any trans-acting factor. The functional significance of this novel structural motif, which is also conserved in dbpA, FRG Y1, and FRG Y2, will be discussed.

Derepression of beta-lactamase (penicillinase in Bacillus cereus by peptidoglycans.

In Bacillus cereus 569 a cellular inducer of beta-lactamase was isolated which has the same constituents and basic structure as the soluble peptidoglycan found in sporulation, extracts from spores, and germination extracts, and which was previously called "spore-peptide." The material has been extensively purified and characterized. Two acid-soluble, high-molecular-weight peptidoglycan fractions containing muramic acid, glucosamine, diaminopimelic acid, d-aspartate, and d- and l-alanine, -lysine, -glycine, and -glutamate, distinguishable on the basis of size and different amino acid to amino sugar ratios, have been found to be responsible for the observed induction. Both fractions are capable of inducing high levels of beta-lactamase in concentrations lower than those of benzyl penicillin required for optimal induction. Several experiments also suggest that it is the accumulation of such soluble peptidoglycan in penicillin-treated cells which leads to induction of beta-lactamase and not the penicillin itself. The "spore-peptide" inducer becomes available during sporulation, and endogenous derepression of beta-lactamase activity occurs simultaneously. Such derepression also occurs in a strain of B. cereus very sensitive to penicillin and in which both uninduced as well as "spore-peptide"-induced beta-lactamase is a small fraction of that produced by the typical penicillinase producer. These results suggest that beta-lactamase in B. cereus functions in cell wall metabolism during sporulation.